The decomposition of organic hydroperoxides as catalyzed by chloroperoxidase was investigated with electron spin resonance (ESR). Tertiary peroxyl radicals were directly detected from incubations of tert-butyl hydroperoxide or cumene hydroperoxide with chloroperoxidase at pH 6.4. Peroxyl , alkoxyl , and carbon-centered free radicals from tertiary hydroperoxide/chloroperoxidase systems were successfully trapped by the spin trap 5,5-dimethyl-1-pyrroline N-oxide, whereas alkoxyl radicals were not detected in the ethyl hydroperoxide/chloroperoxidase system. The classical peroxidase mechanism is proposed to describe the formation of peroxyl radicals. In the case of tertiary peroxyl radicals, their subsequent self-reactions result in the formation of alkoxyl free radicals and molecular oxygen. In the case of the primary ethyl peroxyl radicals, decay through the Russell pathway forms molecular oxygen. Evidence for the production of singlet molecular oxygen was found. The lipid peroxyl radicals from the peroxidation of polyunsaturated fatty acids by soybean lipoxygenase were directly detected by the method of rapid mixing, continuous flow ESR. When air-saturated, pH 9.0 borate buffer containing linoleic acid or arachidonic acid was mixed with lipoxygenase, fatty acid-derived peroxyl free radicals were readily detected with a characteristic g-value of 2.014. Fatty acids without at least two double bonds, e.g., steric acid and oleic acid, did not give the corresponding peroxyl free radicals, suggesting that the formation of a bisallylic carbon-centered radical preceded that of peroxyl radical. The doublet feature of the arachidonate peroxyl spectrum was proven (by selective deuteration) to be a hyperfine coupling due to a gamma-hydrogen, which originated as a vinylic hydrogen of arachidonate.